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В статье использованы материалы сайта http://molbiol.
ru
MORPHOFUNCTIONAL CHANGES IN THE CORTEX OF THE ADRENAL GLANDS OF RATS IN CASE OF CHRONIC ALCOHOLIZATION AND CORRECTION BY
ANTIOXIDANTS
Kvaratsheliya A.G.
Candidate of Biological Sciences, Senior Lecturer Voronezh State Medical University named after N.N. Burdenko, Voronezh, Russia
Klochkova S.V. Doctor of Medicine, Professor First Moscow State Medical University named after I.M. Sechenov, Moscow, Russia
Alekseeva N.T. Doctor of Medicine, Docent Voronezh State Medical University named after N.N. Burdenko, Voronezh, Russia
ABSTRACT
Changes in the parameters of the cytoplasm and nuclei of the cells of the cortex of the adrenal glands as well as nuclear-cytoplasmic ratio are shown in an experiment on chronically alcoholized rats. The content of cholesterol and lipids are studied. It was found that the metabolic correction with a-tocopherol has a positive impact on the morphological and functional state of the cells in the adrenal glands in case of chronic alcohol intoxication.
Keywords: adrenal glands, cortex, corticosterocytes, alcohol intoxication, antioxidant.
Introduction
In modern biology and medicine it becomes very important to study the impact of chronic alcohol intoxication on the body. Especially, more attention is given to the studies of the morphological and functional conditions of the endocrine system since it regulates vital processes in the human body [3, p. 320; 15, p. 2176-2183]. It was found that in case of alcohol intoxication morphological changes occur in the cortex of the adrenal glands (AG) [7, p. 91; 9, p. 58]. Being said that AG themselves affect the process of intoxication by accelerating the removal of ethanol [10, p. 576]. Extent and duration of these changes depend on the dose and concentration of ethanol [5, p. 26; 9, p. 58].
Alcohol damage to AG is a direct result of long-term use of ethanol [5, p. 26; 8, p. 226-228]. The damages and their manifestations are caused by acetaldehyde and other products of the oxidation of ethanol. Nowadays the research on the impact of chronic alcohol intoxication on the cortex of AG is relevant since the hormones of the cortex of AG are directly involved in the protective-adaptive reactions of the organism and ensure homeostasis.
According to several studies, changes of the structure and function of membranes are the basement for the dysfunction of organs and tissues in many diseases. One of the reasons that cause significant damage to the plasma membrane and membranes of the intracellular organelles of cells is an activation
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of lipid peroxidation [2, p. 20-21; 4, p. 425-427]. The action of a-tocopherol and synthetic antioxidants in the therapeutic doses results in a decrease of the level of lipid peroxidation in blood plasma and homogenates of the liver in animals [6, p. 25; 13, p. 207-213]. Alpha-tocopherol proved to inhibit the process of apoptosis [11, p. 1145-1155; 12, p. 7-10; 14, p. 693; 16, p. 163]. In this connection it is of considerable interest to study the modifying effect of antioxidants such as a-tocopherol on the structural and functional organization of AG [1, p. 82-87].
Material and Methods
The experiment was carried out on white outbred male rats weighing 200-210 g. Animals were divided into 2 control and 6 experimental groups. The first control group of rats was kept in the ordinary vivary conditions. Second control group of animals received the intraperitoneal injections of a-tocopherol once a day for 20 days at the rate of 0.1 ml per 100 g of body weight of the animal.
Animals of the experimental group received alcohol as the sole source of enteral fluid intake in free grant mode. The first group consisted of animals that consumed 15% ethanol for 60 days. Material was received on the 60th day. The second group included animals that consumed 15% ethanol for 40 days. From the 41st day animals were injected with 20% -tocopherol intraperitoneally. Material was taken on the 60th day. Animals of the third group consumed ethanol for 40 days. On the day 41 for the animals of this group alcohol was changed to water and the intraperitoneal administration of 20% ^-tocopherol was given for 20 days. The material was taken on the 60th day. The fourth group of rats consumed ethanol for 80 days. The material was received on the 80th day. Animals of the fifth group consumed 15% ethanol for 80 days. From the 61st to 80th day the animals were injected intraperitoneally with 20% solution of ^-tocopherol. Material was taken on the 80th day. The sixth group was allowed to consume 15% ethanol for 60 days. Starting from the day 61 for animals in this group alcohol was replaced with water and the intraperitoneal administration of 20% ^-tocopherol was given for the remaining 20 days. The material was taken on the 80th day.
For morphological studies of AG one part of the excised tissue was fixed in 10% neutral formalin and another - in Bouin's fluid for 24 hours. For histochemical study part of the excised material was frozen in petroleum ether using liquid nitrogen. For morphometric analysis performance of karyometric measurements, study of nuclear-cytoplasmic index and the width of the zones of the cortex of AG were done on preparations stained with hematoxylin and eozin. RNK in the nucleolus and cytoplasm of the cells was detected using histochemical reactions by Brashe (1942) in modification of Kurnik. Content of the protein in the nucleus and cytoplasm of corticosterocytes was studied on sections stained with mercuric bichloride-bromphenol blue by D. Mazia, P. Brezer, M. Alfert (1953). The content of total lipids was detected by the color of fat red "O." Cholesterol was determined by a qualitative assessment of its content in the various areas of the cortex of AG in polarized light after the conclusion of the preparation in glycerol-gelatin.
For quantitative assessment of studied histochemical indicators, the original method of densitometric changes based on the estimate of the values of channel L of color space Lab as
a channel containing information about the brightness of the analyzed area was utilized. Statistical analysis of the data was performed using the software package Statistica 6.1.
Results
Analysis of the cortex of AG showed that the width of zona glomerulosa decreased in the groups of animals that consumed alcohol during 60 and 80 days and did not have the metabolic correction by antioxidants. This was more evident in the fourth group of experimental animals where the changes were statistically significant compared to those of the control groups. The progressive increase in the blurring of boundaries between zona glomerulosa and zona fasciculata in these groups of animals was noted. In the groups of animals treated with a-tocopherol we observed clearly pronounced dependence of the width of the zones on the duration of alcohol intake. It should be noted that in the sixth group of animals that was treated with alcohol and rested for 20 days the width of the zona glomerulosa approached the parameters of the vivary control group.
The study of the dynamics of nuclear cytoplasmic index showed quite pronounced and statistically significant changes in I and IV groups of animals. In I group compared to control groups nuclear cytoplasmic index increased; whereas, in IV group nuclear cytoplasmic index decreased.
In the groups that received the metabolic correction inverse dynamic was seen compared to groups I and IV. At the same time in the group III the width of the zona glomerulosa of the cortex, nuclear cytoplasmic index and parameters of the total protein of cytoplasm approached the level of those parameters of the control group. Changing the protein content in the cytoplasm in this case can be regarded as a manifestation of the stress response that not even reaches the extent of disadaptation.
In all experimental groups an increase in the width of the zona fasciculata was observed. The most pronounced changes in the data were seen in group IV and V. While there were significant differences in the width of the zona fasciculata with respect to vivary control of I and IV experimental groups. In relation to the control group of animals treated with a-tocopherol significant differences have not been identified. It was noted that there are almost the same values of the analyzed parameters in relation to those of the control in the VI group. When comparing the duration of the exogenous intake of alcohol in the absence of metabolic correction, we did not see statistically significant dependence on volume of nucleus. At the same time, there was a statistically significant decrease in nuclear cytoplasmic index in IV group compared to I group.
In groups of animals treated with antioxidant (II, III and V), a statistically significant increase in volume of the nucleus has been identified. When analyzing nuclear cytoplasmic index in all groups compared to control, statistically significant differences were not observed. We noticed that by decreasing the period of alcohol intake, nuclear cytoplasmic index was lowered because of the increase of the volume of nucleus. When comparing with the histochemical data, changes of morphometric parameters showed that in the groups of animals receiving limited amount of alcohol multidirectional changes in total protein content in the cytoplasm and the nucleus of cells took place. In case of the increase in the duration of the use of alcohol, a decrease of the protein concentration in the nucleus and increase in its
concentration in cytoplasm were observed. A strong correlation between the duration of alcohol intake, protein concentration and nuclear cytoplasmic index were seen. Comparison of the optical density of RNA and protein values in the cytoplasm and nucleus of cells showed that the increase in RNA content was accompanied by a simultaneous increase in the volume of the nuclei. In I group these changes were less pronounced than in the group IV. The value of regressional coefficients indicates the phase changes with a tendency to deplete at a long term of alcohol intake.
Determined changes to some extent were confirmed by the relevance of the phase nature of the dynamics of the parameters of the optical density of RNA and total protein in the nucleus and cytoplasm. In groups of animals, which were treated with antioxidants, these changes were less evident. When doing the analysis of the dynamics of the histochemical indicators depending on the duration of alcohol intake, U-shaped nature of the resulting curves was determined: in case of the least time of the alcohol intake and presence of the rest period during prolonged alcohol exposure, the values of the studied histochemical markers were similar; while, in the chronic administration of ethanol despite antioxidant therapy degree of change approached those parameters that were seen in the absence of metabolic correction. Dynamics of indicators in the zona glomerulosa can be regarded as the primary reaction to stress. At the same time we noted the trends to an increase in the width of the cortex of AG and nuclear cytoplasmic index. Metabolic correction reduced the changes that were identified in I and IV experimental groups.
In the analysis of the morphometric parameters of the zona reticularis, we established that the width of this zone did not change significantly in the animals receiving exogenous alcohol. Wherein there was a tendency to increase in its width in groups I and IV by 10.8 and 11.3% respectively. We also noted an increase in the variability of the indicator in group IV as compared to control and I group.
In groups of animals treated with a-tocopherol, we observed statistically significant differences in the width of the zona reticularis (II, V and VI groups). In group III that had the lowest duration of alcohol intake statistically significant differences were not found. It should be noted that in all groups a trend to increase in the width of this zone was observed and reached its maximum in the V group. The simulation results showed a relative increase in the width of the zone by increasing the cross sectional area of the cytoplasm. We noted that a decrease in the volume of the nucleus was accompanied by the increase in the duration of alcohol administration. In the VI group of animals that received a-tocopherol and had a period of rest we identified significant difference in nuclear cytoplasmic index compared to that in the V group.
When comparing the data of karyometry, nuclear cytoplasmic index and protein content of the nucleus, we found that the increase in the duration of exogenous alcohol intake was accompanied by a decrease of the optical density of protein in the nucleus, which had close cross-sections. Thus, a comparative study of histochemical and morphometric analysis of the data suggests an increase of degenerative processes in the background of karyopyknosis in case of chronic administration of ethanol.
In groups of animals that had the metabolic correction modeling of the dynamics of morphometric and histochemical parameters showed a close regressional dependence of the size of the nucleus, nuclear cytoplasmic index and histochemical markers - total protein and RNA of nucleolus - on the duration of ethanol consumption. Closest to the control group parameters were seen in the animals of groups III and VI, i.e. with the least duration of consumption of ethanol (III group) and a rest period from its consumption. Study of the results of modeling of the width of the zona reticularis, nuclear cytoplasmic index and optical density of the protein in the cytoplasm of cells showed a pronounced statistically significant difference in the groups of animals treated isolatedly with alcohol (I and IV). It is noteworthy that flattening of resulting curves and surfaces was seen with the increase in the duration of the use of alcohol. In this case, the increase in nuclear cytoplasmic index corresponded to a proportional increase in the optical density of the protein in the cytoplasm, which probably reflects the process of adaptation to stressful exogenous metabolic factors - ethanol.
Highly expressed degree of change of lipid metabolism during chronic administration of alcohol is accompanied by activation of the processes of lipogenesis and cholesterol synthesis. It was found that increase in the duration of alcohol intake in I and IV groups leads to changes in the nature of the distribution of cholesterol granules in comparison with the control group. In the control group a typical pulverized uniform distribution of optically active fills in corticosterocytes was seen using polarized light. At the same time, in I and IV groups we noted a trend toward grouping of mentioned above optical phenomena with the formation of microblocks that predominantly localized in the corticosterocytes of zona granulosa and fasciculata. Considering the previously experienced histochemical and morphometric changes, one can assume that in this case we are faced with the accumulation of metabolic precursors of hormones, which because of the broken synthetic processes in the cell in case of chronic alcohol intake, did not undergo metabolic changes due to the negative dynamics of biochemical processes. In the groups that received metabolic correction, similar changes were revealed; however, lesser period of time of exogenous alcohol intake and the presence of the rest period brought closer to a qualitative picture of the distribution of optical phenomena associated with cholesterol in the control group.
An increase in the number of inclusions of cholesterol in the form of the agglomeration of granules in groups III and VI as compared to controls and to the group of animals under long alcoholization was noted. When comparing between the groups III and IV a less pronounced tendency to agglomeration and to increase in cholesterol inclusions in animals of III group was observed, which may be regarded as more intensive incorporation of cholesterol metabolism as a precursor of steroid hormones in case of long term alcoholization.
Detection of total lipids showed a marked change in their distribution areas of the cortex in all experimental animals. In the I and IV groups we observed the zones of reduction of the lipid content on the boundary of zona granulosa and fasciculata. Perhaps observed changes reveal a violation of reparative processes in the cortex of AG by reducing the
functional activity of the zones of the growth layer. It should also be noted that a comparison of the localization of these changes are consistent with the identified blurring of the boundaries of the zona granulosa and fasciculata.
Conclusion
Thus, the identified changes, which reflect the quantitative characteristics of the cells of different areas of the cortex of AG, are in close functional relationship with the biochemical parameters that determine the activity of protein synthesis. The observed structural and functional changes and parameters of protein metabolism are closely correlated with changes in lipid metabolism, in particular with the cholesterol metabolism. Metabolic correction using a-tocopherol has a positive impact on the morphological and functional state of the cells of AG in developed experimental model.
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